Centrosomes (or spindle pole bodies (SPBs) in budding yeast) are microtubule organizing centers that nucleate microtubules in interphase and establish the mitotic spindle during mitosis. The SPB/centrosome organizes the bipolar mitotic spindle to capture duplicated chromosomes and segregate them to the new daughter cells. Proper regulation of centrosome duplication and function is necessary to maintain a normal diploid genome, and aberrations in centrosome structure and/or copy number are often correlated with loss of genomic integrity in many types of tumors. One important method of SPB/centrosomal regulation is through protein phosphorylation. Several phosphorylation sites have previously been identified on SPB proteins, and mutations of some of these phosphorylation sites leads to defects in SPB assembly or microtubule nucleation and spindle organization. The project consists of two major parts. First is to establish a comprehensive inventory of phosphorylation events found on the 18 core SPB proteins in yeast, using mass spectrometry to analyze enriched, intact SPBs. To date, 85 novel phosphorylation sites have been identified in this project. Second, the identified phosphorylation events will be analyzed to determine which are important in regulatory mechanisms of SPB/centrosome function. During this fellowship period, research will be focused specifically on the phosphorylation-dependent regulation of the essential and well-conserved gamma-tubulin sub-complex, which functions in microtubule nucleation. Phosphorylation sites identified within the gammatubulin complex will be prioritized and chosen for mutational analysis on the basis of sequence conservation, results from previous mutational studies, and on whether the phosphorylation event is regulated during the cell cycle. Overall, this project will expand our understanding of centrosome regulation and the role of phospho-regulation in maintaining genomic integrity, commonly lost during tumorigenesis. Public Health Relevance: The centrosome is a large cellular protein complex that organizes the machinery used to separate chromosomes between newly divided cells, and defects in centrosome assembly and/or function can result in chromosome division errors and tumor formation. One manner of regulating centrosome function is through chemical modification by the addition of phosphate groups (called phosphorylation). The proposed project will identify these phosphorylation sites within the centrosome and study how altered phosphorylation states can cause chromosome segregation defects in the cell. [unreadable] [unreadable] [unreadable]